Epithelial organization and cyst lumen expansionrequire efficient Sec13–Sec31-driven secretion

Anna K. Townley1,*, Katy Schmidt1,*,`, Lorna Hodgson1 and David J. Stephens1,§

1Cell Biology Laboratories, School of Biochemistry, Medical Sciences Building, University of Bristol, University Walk, Bristol, BS8 1TD, UK

*These authors contributed equally to this work`Present address: Max F. Perutz Laboratories Dr. Bohr Gasse 9/3 1030 Wien, Austria§Author for correspondence (david.stephens@bristol.ac.uk)

Accepted 26 October 2011Journal of Cell Science 125, 673–684� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.091355

SummaryEpithelial morphogenesis is directed by interactions with the underlying extracellular matrix. Secretion of collagen and other matrixcomponents requires efficient coat complex II (COPII) vesicle formation at the endoplasmic reticulum. Here, we show that suppressionof the outer layer COPII component, Sec13, in zebrafish embryos results in a disorganized gut epithelium. In human intestinal epithelial

cells (Caco-2), Sec13 depletion causes defective epithelial polarity and organization on permeable supports. Defects are seen in theability of cells to adhere to the substrate, form a monolayer and form intercellular junctions. When embedded in a three-dimensionalmatrix, Sec13-depleted Caco-2 cells form cysts but, unlike controls, are defective in lumen expansion. Incorporation of primary

fibroblasts within the three-dimensional culture substantially restores normal morphogenesis. We conclude that efficient COPII-dependent secretion, notably assembly of Sec13–Sec31, is required to drive epithelial morphogenesis in both two- and three-dimensionalcultures in vitro, as well as in vivo. Our results provide insight into the role of COPII in epithelial morphogenesis and have implicationsfor the interpretation of epithelial polarity and organization assays in cell culture.

Key words: COPII, ER export, Epithelial organization

IntroductionEpithelial morphogenesis requires formation of cell–substrate

contacts to enable polarization of cells. Cell–substrate contacts are

defined as interactions between cells and a specialized extracellular

matrix (ECM), the basal lamina, which consists mainly of collagen

and laminin. The ECM is generally secreted by epithelial cells.

Elucidating the molecular mechanisms underlying the interactions

between epithelial cells and basal lamina, culminating in formation

of an epithelial sheet or a tube of cells, is crucial for our

understanding of development, organ function, and the onset and

progression of disease (reviewed by Bryant and Mostov, 2008).

Membrane traffic is essential to establish and maintain cell polarity

through apical and basal targeting of proteins as well as through

directed recycling of components between these membrane domains

(Mellman and Nelson, 2008). Increasing evidence shows that

directed secretion of ECM is a key requirement in establishment of

polarity. Membrane trafficking through the early secretory pathway

has been implicated in tube or lumen formation in several systems in

vivo (Tsarouhas et al., 2007; Grieder et al., 2008; Jayaram et al.,

2008; Norum et al., 2010). For example, the Drosophila mutants

haunted and ghost show defects in epithelial polarity as well as in

secretion into the luminal matrix of the trachea and cuticle

deposition. The haunted and ghost genes encode the coat complex

II (COPII) proteins Sec23 and Sec24, respectively (Norum et al.,

2010). The COPII component Sar1 has been shown to be required

for luminal matrix assembly and tube expansion of Drosophila

trachea (Tsarouhas et al., 2007). More recently, Sec24 has been

shown to be essential for lumen expansion in tracheal development

in a cell autonomous manner (Forster et al., 2010). Extensive

secretion of atypically large cargo is also essential for cuticle

formation, which relies on sar1, sec23 and sec13 function (Abrams

and Andrew, 2005). In addition, it has been shown that expression of

COPII components is upregulated during development of the

Drosophila salivary gland (Abrams and Andrew, 2005), a highly

tubulated organ that has a high secretory load.

The COPII coat (Barlowe et al., 1994) directs cargo selection

and budding of transport carriers from the ER membrane

(reviewed by Hughes and Stephens, 2008). COPII assembly is

triggered by Sec12-dependent activation of the small GTPase

Sar1 (d’Enfert et al., 1991), which recruits the heterodimeric

major cargo selection module Sec23–Sec24 (Kuehn et al., 1998)

to form the pre-budding complex. These pre-budding complexes

subsequently recruit an additional layer of the COPII vesicle

coat, Sec13–Sec31, which enhances GTP hydrolysis on Sar1 and

completes budding of the vesicles (Salama et al., 1997; Antonny

et al., 2001; Townley et al., 2008). COPII vesicles formed in

vitro are typically 60–80 nm in size (Matsuoka et al., 1998;

Antonny et al., 2003). The cages that spontaneously assemble

from purified Sec13–Sec31 (Stagg et al., 2006) and those that

are seen in or purified from cells (Aridor et al., 1999; Matsuoka

et al., 2001) are also 60 nm in size. This presents an inherent

problem for the packaging of large secretory cargo and,

consequently, for characteristic components of the basal

lamina, notably linear rod-like molecules such as fibrillar

procollagen type I (,300 nm) (Canty and Kadler, 2005), and

potentially for other ECM molecules, e.g. laminin (up to

120 nm) (Beck et al., 1990) and perlecan (up to 200 nm)

(Farach-Carson and Carson, 2007).

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We recently established that RNA interference (RNAi)-

mediated suppression of Sec13 results in depletion of the entireouter layer of the COPII vesicle coat complex and causes aselective defect in collagen secretion (Townley et al., 2008) in

development of the craniofacial skeleton but probably also ofother large ECM molecules (Townley and Stephens, 2009).Because of their shape and size, large cargos including theseECM components are more likely to rely on a strengthened and

persistent vesicle coat than small soluble molecules would be.This implies a role for the outer COPII coat, Sec13–Sec31, inscaffolding and stabilizing transport carriers containing atypically

large cargo (Fromme and Schekman, 2005; Townley andStephens, 2009). A current model proposes that export of largecargo requires highly efficient coupling between the inner COPII

layer, Sar1 with Sec23–Sec24, and the COPII outer layer, Sec13–Sec31 (Schmidt and Stephens, 2010). Mutation of Sec23A resultsin inefficient assembly of the full COPII coat, with the resulting

defects in collagen secretion from chondrocytes causing cranio-lenticulo-sutural dysplasia (Boyadjiev et al., 2006; Bi et al., 2007;Fromme et al., 2007).

In order to determine whether Sec13–Sec31 is generally

required for the transport of large cargo as opposed to a restrictedaction in chondrogenesis, we examined the effects of knockdownof Sec13–Sec31 in gut morphogenesis in zebrafish embryos and

an intestinal epithelial cell culture system. Establishment of abasal lamina coincides with generation and polarization of anepithelial cell layer, which provides an ideal read-out foreffective secretion of large cargo both, in vitro and in vivo.

The most notable defect observed in the intestine of Sec13–Sec31-depleted zebrafish embryos was the disorganization of theintestinal epithelium along with a restricted gut lumen. We

therefore sought to define the functional necessity of Sec13–Sec31 in epithelial polarity and organization of Caco-2 (humancolon cancer) cells in vitro. Here, we show that on-going efficient

COPII-dependent secretion is essential during epithelialmorphogenesis in vitro and in vivo. These data demonstrate theimportance of careful interpretation of experimental outcomes

from two- and three-dimensional (2D and 3D, respectively)epithelial cell culture systems in which defined phenotypescould arise from defects in secretion rather than the primaryexperimental manipulation.

ResultsDefective lumen expansion in zebrafish Sec13 morphants

We analyzed the organization of intestinal epithelia in Sec13

morphant zebrafish embryos in order to define the role ofSec13–Sec31-dependent transport for establishment of the basallamina in intestinal morphogenesis. Two translation-blockingmorpholino oligonucleotides targeted against Sec13 were used

(Sec13-1 and Sec13-2); the phenotypes were indistinguishableusing either of them (see also Townley et al., 2008). Results fromonly one morpholino (Sec13-2) are shown in Fig. 1. The

intestinal phenotype of all zebrafish included in this study wasdefined by showing pectoral fin defects, as described previously(Townley et al., 2008). The intestinal epithelium of Sec13

morphants was disorganized compared with age-matchedcontrols on sections taken from the same level along thecranio–caudal length of the embryos. Sec13-suppressed

zebrafish showed inefficient monolayer formation anddramatically restricted intestinal lumen expansion (Fig. 1A),suggesting that the polarization of epithelial cells might be

affected by the reduction in Sec13 levels. To evaluate the cellular

phenotype of the Sec13 morphant gut lining, we conducted

electron microscopy on thin sections from the same samples.

Fig. 1. Disorganization and limited expansion of the zebrafish gut lumen

following Sec13 suppression. (A) Transverse sections through the gut of

5 dpf zebrafish embryos injected with control (left) or Sec13 morpholinos

(right). Sections (1 mm) were taken from a comparable level along the cranio–

caudal axis of age-matched embryos. Note the limited expansion of the lumen

of the gut and disorganization of the gut epithelium in Sec13 morphants.

(B) The intracellular organization of the ER–Golgi interface is disrupted in

morphant epithelial cells. Suppression of Sec13-induced distension of the ER

and accumulation of budding profiles (see insets for second example).

(C) Sec13-depleted intestinal epithelial cells are attached to surrounding cells

but a clear basal lamina (indicated by dotted lines, compare with control) is

missing. (D) TEM of intestinal epithelial cells reveals that the microvilli seam

was intact in Sec13 morphants but cell monolayer formation was

disorganized, as evident from the diverse range of surface areas of the cells

reaching the lumen. Total of three embryos analyzed for each oligo used. The

intestinal phenotype of all of zebrafish included in this study was defined by

showing pectoral fin defects as published previously (Townley et al., 2008).

BL, basal lamina; E, epithelial cells; I, intestinal lining; N, nucleus. Scale

bars: 0.5 mm (B,C); 1 mm (D).

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Indeed, the structure of the ER and Golgi were perturbed in

Sec13 morphants (Fig. 1B), consistent with a trafficking defect.

The apical surface showed a developing microvilli seam in

controls as well as in morphant fish, indicating that overall cell

morphology was not completely disrupted. The failure to polarize

epithelial cells and generate a regular gut epithelium suggested a

basolateral trafficking defect. Indeed, Sec13 suppression coincided

with a disrupted and reduced basal lamina in morphant embryos

(Fig. 1C, compare dotted lines). Collagen IV, of which most of the

basal lamina is comprised, was the most likely candidate for a

transport defect. The uneven contribution of single cells to the

apical surface in Sec13 morphants (Fig. 1D) further indicated

compromised monolayer formation of the gut epithelium, as seen

in the histological sections (Fig. 1A). This defect did not arise

from gross differences in cell size (Fig. 1, compare D with E; the

irregular contrasting in Fig. 1D was due to a sectioning artefact).

Sec13–Sec31 is required for epithelial morphogenesis

in vitro

In order to define the mechanistic requirements for high

efficiency COPII-dependent secretion we suppressed Sec13 in

Caco-2 cells, a human epithelial colon cancer cell line. Because

Caco-2 cells require 2 weeks to develop polarity on permeable

supports and to form cysts when embedded in 3D matrix, we used

lentiviral transduction to stably suppress expression of Sec13.

Immunoblotting was used to confirm suppression of expression

of both Sec13 and Sec31A in stable cell lines (Fig. 2A). We have

previously shown that depletion of Sec13 results in concomitant

suppression of Sec31A (Townley et al., 2008), resulting in highly

effective reduction in the amount of the outer layer of the COPII

coat. We imaged epithelial organization in polarized monolayers

of control and Sec13-suppressed Caco-2 cells grown on

permeable supports using confocal microscopy to view cells in

X–Z view (i.e. parallel to the filter support). In control cultures, a

single monolayer of cells was formed but in Sec13-depleted cells,

the monolayer organization was lost, with cells growing over one

another to form a multilayered sheet of cells (Fig. 2B). This

disorganization was particularly obvious following 3D rendering

of cell nuclei (Fig. 2C, each nucleus is shown in a different

colour). Cells failed to grow as a single cell layer, with large

areas of the filter showing cell layers of two, three and even four

cell depths. Electron microscopy of Caco-2 monolayers showed

that Sec13 suppression caused extensive disorganization of

intercellular junctions with extended areas of interdigitated

plasma membrane compared with control cells treated with

scrambled shRNA (Fig. 2D, arrows).

We then used scanning electron microscopy (SEM) and

transmission electron microscopy (TEM) to define the defects

in monolayer organization of Caco-2 cells grown on permeable

supports in more detail. No defects were seen in the ability of

cells formation of microvilli at the apical surface following

Sec13-Sec31 suppression (Fig. 3, compare B with A). Monolayer

Fig. 2. Stable suppression of Sec13

expression in Caco-2 cells.

(A) Immunoblotting of lysates from cells

transduced with lentiviral constructs as

indicated. Lysates of cells stably expressing

shRNA were immunoblotted for Sec13,

Sec31A and a-tubulin as indicated.

Molecular markers are shown in kDa. The

asterisk marks a nonspecific band detected

by the antibody. (B) X–Z reconstructions of

DAPI-labelled nuclei of epithelial

monolayers grown for 14 days on a

permeable support from control or Sec13-1

Caco-2 cells. (C) Automatic detection of cell

nuclei and pseudo-colouring highlights the

disorganization of the epithelial layer

following suppression of Sec13. (D) TEM of

polarized Caco-2 monolayer cultures from

control and Sec13-1-suppressed cells. Note

the extensive interdigitation of intercellular

junctions on suppression of Sec13 (arrows).

Scale bars: 2 mm.

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organization was clearly affected (Fig. 3C, as seen in Fig. 2B,C).

Attachment to the filter support was also reduced, as judged by

the proximity of the basal membrane to the filter in electron

microscopy sections (Fig. 3D, dashed lines). The intercellular

junctions between cells were strongly affected by Sec13

depletion (Fig. 3E, arrows indicate the extensive gaps evident

between cells depleted of Sec13), and clear defects in

intracellular membrane traffic were evident (Fig. 3F, note

distension of the ER, arrow). These trafficking defects

recapitulate those we have described previously following

depletion of Sec13 in HeLa and fibroblast cells (Townley et al.,

2008). The data from Caco-2 cells showed that the situation in

vitro largely resembles that in vivo.

We then sought to determine whether this phenotype could be

caused cell-autonomously by a failure of the intestinal epithelial

cells to secrete specific matrix components, rather than a

generalized lack of ECM, which could occur in zebrafish

morphants where all cells, not just intestinal epithelial cells, are

suppressed for Sec13. In situ, intestinal epithelial cells grow

on a basement membrane largely composed of collagen IV

and laminin-1 (Timpl, 1996). The attachment to this basement

membrane directs polarization and epithelial organization of

intestinal cells. On the basis of our initial results we hypothesized

that the defects in monolayer organization in Sec13-depleted

Caco-2 cells resulted from defective ECM secretion or assembly

(Townley et al., 2008; Townley and Stephens, 2009). We

therefore aimed to replace the putatively incomplete ECM by

coating the permeable supports with collagen IV prior to

seeding cells. Caco-2 cells grown on collagen-IV-coated filters

for 14 days showed marked improvements in monolayer

Fig. 3. Electron microscopy of epithelial

organization in Sec13-depleted Caco-2 cells.

(A,B) SEM of microvilli on the apical surface of Caco-

2 monolayers grown for 14 days on transwell filters:

(A) control cells, (B) Sec13-depleted cells.

(C–F) Polarized Caco-2 cells stably expressing control

or Sec13 shRNAs as indicated were examined for

(C) polarity (i.e. the formation of monolayers),

(D) attachment to the substratum (dotted lines indicate

basal cell surface), (E) intercellular adhesion (cell–cell

attachment indicated by arrows) and (F) ER–Golgi

membrane organization. (G–J) Cells were also

examined by TEM following growth for 14 days on

collagen-IV-coated permeable supports (G, polarity;

H, attachment; I, adhesion; J, ER and Golgi membrane

traffic). Two independent samples were analyzed for

each condition. N, nuclei. Scale bars: 2 mm (A–C);

1 mm (D,E); 0.5 mm (F).

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organization (Fig. 3G), attachment to these filters (Fig. 3H) and

intercellular adhesion (Fig. 3I, arrows). Notably, no difference interms of enlarged ER (Fig. 3J, arrow) was seen between Sec13-depleted cells grown in the presence or absence of collagen IV,

indicating that the trafficking defect could not be restoredby replacing a putatively missing ECM component. Ourinterpretation of these data is that exogenously added collagenIV improves cell–matrix adhesion and polarization of the cells

and, hence, partially compensates for the failure to transport largecargo. By contrast, changes in the ER–Golgi structure and in budformation are associated directly with the loss of Sec13-Sec31

and cannot be rescued by outside-in signalling from the ECM.

COPII-dependent secretion is essential for epithelialmorphogenesis in 3D culture

Because ‘rescue’ of the phenotype by collagen IV alone wasincomplete, we reasoned that additional secreted components areprobably involved. In order to define whether ECM proteins

might compensate for a secretion defect in Sec13-depleted cells,we embedded cells in 3D matrix because a complete matrix todirect polarization, polarity and organization would be providedexogenously. Engelbreth–Holm–Swarm (EHS)-tumour-derived

matrix (from mice) is widely used for 3D cell culture andcomprises primarily collagen IV, entactin, perlecan and laminin(Grant et al., 1985). The laminin is predominantly laminin-1,

including the a1, b1 and c1 subunits, i.e. the essential form oflaminin for correct Caco-2 polarity and organization (DeArcangelis et al., 1996). Caco-2 cells form cysts when

embedded in 3D EHS-derived matrix such as Matrigel orGeltrex (Ivanov et al., 2008; Jaffe et al., 2008). We anticipatedthat embedding Sec13-depleted cells in such matrix would

support the formation of cysts indistinguishable from those ofcontrol cells.

Control cells formed spherical 3D cysts as expected (Fig. 4A,control). Apical–basolateral polarity was established and lumen

expansion was evident (Ivanov et al., 2008; Jaffe et al., 2008).Comparing only cyst-like formations, the localization of polaritymarkers b-catenin (Fig. 4A) or epithelial-specific antigen (ESA;

Fig. 4B) to the basolateral membrane was similar in bothcontrol and Sec13-suppressed cysts. Some increased laterallabelling of b-catenin was evident (Fig. 4A), as was someenhanced intracellular labelling of ESA (Fig. 4B). Assembly of

filamentous actin at the apical surface was evident in all cystsformed from Sec13-suppressed cells (Fig. 4A,B). However,Sec13-suppressed cells grew into much smaller cysts with a

small but significant reduction in cell number (Fig. 4C). Mostobviously, Sec13-depleted cysts failed to show efficient lumenexpansion (Fig. 4A,D) and indeed often failed to form any lumen

at all (Fig. 4A, arrows) or formed cysts with multiple lumens(Fig. 4B, arrows). Quantification of lumen size confirmed amarked defect in lumen expansion following Sec13 suppression

(Fig. 4D). Lumen expansion requires fluid filling of the interiorof a cyst. Activation of protein kinase A (PKA) with N6-benzoyl-cAMP (6-Bnz-cAMP), which stimulates PKA-dependent fluidfilling of the lumen (Jaffe et al., 2008) did not enhance lumen

expansion of Sec13-depleted cysts. However, uniformity of thecysts was noticeably improved by addition of 6-Bnz-cAMP andso this was included in all following experiments.

We reasoned that reduced cell number within the cysts and thedisorganization seen could at least in part arise through defectivecontrol of cell division. Recent data showed that depletion of

Cdc42 in Caco-2 cells caused defects in cyst morphogenesiscomparable to the defects seen in our experiments, which were

linked to misalignment of the mitotic spindle axis during cystexpansion (Jaffe et al., 2008). It has been well documented thatthe ECM dictates the orientation of the spindle axis duringmitosis in a variety of systems (Bornens, 2008). A significant

difference in spindle alignment was seen between cysts grownfrom control cells and those grown from Sec13-suppressed cells(Fig. 4E, a-tubulin labelling). Sec13 suppression resulted in a

significant misalignment of spindles in dividing cells grown atthe periphery of cysts (i.e. the direction of cyst expansion)(Fig. 4F). We also noticed significantly more mitotic cells within

the centre of cysts (Fig. 4E). This is consistent with the multi-layering of cells seen in Sec13-depleted cells grown on filters andis in agreement with a failure of signalling from the ECM tospatially direct cell division (Bornens, 2008; Jaffe et al., 2008).

ECM secretion from and deposition by Sec13-suppressed cells

Our data show that exogenously supplied ECM is necessaryto direct polarization and the initial stages of cyst formationin 3D of Sec13-suppressed Caco-2 cells. Because supplying

collagen-IV-rich matrix was not sufficient to complete cystlumen expansion, our data suggested that on-going cellautonomous secretion is required to finalize cyst polarity andorganization. The defects in morphogenesis could result from a

defect in secretion of ECM components that are not supplied byEHS-derived matrix, or from a failure to secrete small, solublefactors.

Our working hypothesis was that Sec13 depletion leads to adefect in deposition of large ECM components such as collagen(Townley et al., 2008), therefore we went on to monitor the

secretion of these macromolecules, initially in 2D cultures. Weused immunofluorescence of ECM deposited by Caco-2 cellsgrown on glass coverslips (following removal of cells). These

experiments showed that Sec13 suppression resulted in reduceddeposition of collagen I and perlecan (Fig. 5A,B). In 3D culture,collagen IV secretion and/or remodelling was not significantlydifferent in control and Sec13-depleted cells (Fig. 5C). The

antibody used was not human-specific and therefore these datareflect the ability of Sec13-depleted cysts to assemble collagenIV derived from the supplied mouse 3D matrix. Cells depleted of

Sec13 also showed a reduction in assembly of laminin-1compared with controls when grown in 3D culture (Fig. 5C,D).The antibody used also detects laminin-1 from EHS-derived

matrix and so these data could reflect either an inability of Sec13-depleted cells to secrete laminin-1, or a failure of cysts toorganize the surrounding laminin network. Immunoblotting ofcell-derived matrix (i.e. that remaining following removal of cells

from the culture dish) with antibodies to different lamininsubunits showed a surprising increase in laminin secretion bySec13-depleted cells (Fig. 5E), demonstrating that the defects

seen in Fig. 5C resulted from a failure to organize the lamininmatrix rather than a failure to secrete laminin. Using 2D gelelectrophoresis of proteins ,50 kDa, we could not detect any

differences in the soluble secretome of control or Sec13-suppressed Caco-2 cells (supplementary material Fig. S1A,B).This was consistent with the lack of detectable defects in the

transport or secretion of small, soluble, freely diffusible proteinsor transmembrane proteins in Sec13-suppressed HeLa cells(Townley et al., 2008) and Caco-2 cells (gp135/podocalyxin;

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data not shown). However, we cannot rule out defects in small

cargo secretion that are undetectable using this approach.

Rescue of cyst lumen expansion by co-culture

with fibroblasts

EHS-derived matrix largely lacks fibrillar collagens (Grant et al.,

1985). Because we found that COPII-dependent secretion of

collagen I is impaired on Sec13 suppression (Townley et al., 2008)

(and this study), we sought to define whether exogenous collagen

type I could rescue lumen expansion in 3D culture. Adding soluble

collagen I to EHS-derived matrix directly did not improve cyst

morphogenesis, but this was probably due to improper fibril

assembly of the purified rat tail protein used. Our data pointed

towards a missing secreted factor that is required for cyst

formation. We chose to determine whether this could be

provided exogenously by co-culturing Sec13-depleted Caco-2

cells with normal human fibroblasts. We incorporated an equal

number of primary human fibroblasts into the 3D culture

(Fig. 6A). In these experiments, control cells grew well and

developed into 3D cysts with fully expanded lumens (Fig. 6A).

Notably, in the presence of fibroblasts, Sec13-depleted cells also

grew into large cysts with enhanced lumen expansion compared

Fig. 4. Sec13 suppression disrupts cyst formation by Caco-2 cells embedded in 3D matrix. (A) Formation of cysts by Caco-2 cells grown for 7 days in 3D

matrix is inhibited by Sec13 suppression. Organization of the cell monolayer is affected compared with controls (DAPI labelling of nuclei), as is expansion of the

central lumen (highlighted by phalloidin labelling of filamentous actin). Cysts are also smaller. (B) Basolateral targeting is unaffected as shown by labelling for

ESA. (C) The number of cells per cyst is also decreased, indicating a cell division defect. (D) Quantification shows that lumen size (lumen area at the point of

maximum cyst width) is significantly decreased on Sec13 suppression (with or without the presence of 6-Bnz). In A–D, n.40 cysts in each of three independent

experiments performed for each experimental condition. Error bars represent s.e.m. P values compare depleted samples with control. (E) Alignment of the mitotic

spindle (tubulin labelling) is perturbed in Sec13-depleted cysts, with frequent mitotic profiles seen in the centre of cysts as well as in the limiting layer.

(F) Quantification of spindle alignment in these cysts. Median values are shown by the horizontal bar within each box; boxes show 25th and 75th percentiles;

whiskers show the spread of the data. In E and F, n530 cysts for each shRNA from three independent experiments. Scale bars: 20 mm.

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with controls (Fig. 6A,B). This increase in cyst size resulted from

an increase in cell number per cyst rather than cell size. Weobserved that the fibroblasts grew as a layer below the cysts and

hence were not visible in the confocal sections shown. We then

used conditioned medium from fibroblasts to define whether asoluble secreted factor would be sufficient to rescue the cyst

expansion defect seen on Sec13 depletion. Fig. 7 shows that

addition of medium from normal dermal fibroblast (NDF) cultures

had no significant effect on growth of control cysts (Fig. 7,compare B with A) and can partially rescue lumen formation in

Sec13-depleted cysts (Fig. 7, compare D with C). These data were

quantified and average lumen sizes (for those cysts with any lumen

expansion) are shown schematically in Fig. 7E–H and graphicallyin Fig. 7I. We sought to define whether this soluble secreted factor

might be collagen I by stably depleting collagen type I a1

(COL1A1) from fibroblasts. However, all fibroblasts transduced

with virus to deplete COL1A1 did not survive in culture, evenwhen grown on a thin coating of Getrex.

DiscussionWe have shown here that Sec13 depletion results in defects of

epithelial morphogenesis in vivo and in vitro. We can largely rescue

epithelial monolayer formation in 2D transwell cultures by addition

of collagen IV. In 3D culture, a collagen-IV-rich matrix is not

sufficient to drive full lumen expansion in 3D. These data highlight

the requirement for efficient COPII-dependent secretion in cyst

morphogenesis in vitro. We can substantially restore cyst generation

of Sec13-depleted Caco-2 cells by adding fibroblasts, or indeed

conditioned medium from fibroblasts, to our 3D culture system. In

both cases, however, the rescue was incomplete. It can therefore not

be excluded that COPII-dependent export from the ER is required to

organize intracellular signalling pathways to direct epithelial

morphogenesis, or that this can be a contributing factor. Equally,

it is possible that the export of one or more small soluble factors

is disturbed such that cyst formation is impaired. However, our

data suggest that this might be unlikely; 2D gel electrophoresis

of the secreted proteome of Sec13-suppressed Caco-2 cells

(supplementary material Fig. S1) revealed no differences in the

secretion of small soluble cargo, similarly to previous work in HeLa

cells (Townley et al., 2008). These data are consistent with the

hypothesis that a stabilized COPII coat is required to direct ER

export of large and bulky cargo such as ECM components (Townley

et al., 2008; Schmidt and Stephens, 2010) but do not rule out a

requirement for the secretion of non-ECM cargoes of any size.

Fig. 5. Deposition and assembly of ECM

surrounding epithelial cysts is impaired

following Sec13 depletion. (A,B) Collagen I

and perlecan deposition by cells grown on glass

coverslips was analyzed by

immunofluorescence microscopy following

removal of cells. B shows quantification of

results. (C,D) Collagen IV assembly around

epithelial cysts grown in 3D matrix is

unaffected by Sec13 suppression. By contrast,

laminin-1 labelling around control cysts covers

a much greater area than that surrounding cysts

formed from Sec13-depleted cells. The

intensity of labelling immediately surrounding

the cyst is also significantly reduced following

Sec13 suppression. Three examples are shown

in C. Quantification of laminin labelling is

shown in D. Median values are shown by the

horizontal bar within each box; boxes show

25th and 75th percentiles; whiskers show the

spread of the data. n526 cysts total from three

independent experiments. (E) Immunoblotting

of cell-derived matrices. Cells were grown for

10 days in tissue culture dishes with ascorbic

acid feeding. Cells were denuded and the

remaining matrix was removed and added to

sample buffer. Samples were immunoblotted

with the following antibodies: laminin c-2,

laminin b-3 and rabbit polyclonal antibody

against laminin (which recognises A chain, B1

chain and B2 chain). Molecular mass is

indicated in KDa. Scale bars: 20 mm.

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Because normal ER morphology was not restored by

supplementing the ECM with fibroblasts, impaired COPII

budding at the ER following Sec13 suppression does not

appear to result from a failure of ECM-dependent outside-in

signalling. We favour a model based on a selective defect in

secretion and assembly of ECM. Attempts to test the requirement

for COL1A1 secretion by fibroblasts in our co-culture system

were not possible. COL1A1 suppression in fibroblasts resulted in

loss of adhesion to plastic dishes and, consequently, we were

unable to amplify these cells for co-culture with Sec13-depleted

Caco-2 cells.

Although our data show that ECM secretion is dependent on

the outer layer of the COPII coat, they do not preclude the

possibility that ECM export from the ER, although COPII-dependent, might not in fact occur in COPII-coated carriers

(Mironov et al., 2003). Such a model implies that the role ofCOPII is in trafficking of accessory factors that are required forcargo export, such as tethers and SNAREs. Our current favouredmodel is that Sec13–Sec31 encapsulates nascent vesicle buds

containing fibrillar collagen and large cargo as they emerge fromthe ER (Stagg et al., 2008; Schmidt and Stephens, 2010). Ourdata are consistent with models in which reduced Sec13–Sec31

causes a failure to scaffold the formation of such carriers.Notably, recent data have shown that Sec13–Sec31 can formtubular structures in vitro that might serve as containers for

fibrillar collagen in vivo (O’Donnell et al., 2010). Whether thisoccurs via a receptor-mediated mechanism as has been proposedfor TANGO1-mediated export of collagen (Saito et al., 2009;Wilson et al., 2011), or simply due to a geometric requirement for

ordered Sec13–Sec31 assembly around larger export containers(Stagg et al., 2008), or even a combination of the two, is aquestion for future research. In further support of the notion of

encapsulation of collagen within a COPII-coated carrier, Aridorand colleagues showed recently that a mutant form of Sar1 (withkey substitutions in a conserved C-terminal loop) did not affect

recruitment of other COPII proteins, nor Sar1 activation, but didinhibit oligomerization of Sar1 and type I procollagen exportfrom the ER (Long et al., 2010). The authors propose that

deregulation of membrane constriction is responsible, such thatthe mutant form of Sar1 indirectly prevents entry of procollageninto Sar1-coated tubules.

A dynamic interplay between both epithelial and surrounding

cells in terms of ECM deposition and remodelling is certainlynecessary to direct morphogenesis in vivo (Kedinger et al., 1998;Simon-Assmann et al., 1998; O’Brien et al., 2001; Martin-

Belmonte et al., 2008). The difference in ECM compositionsurrounding Sec13-depleted cysts could be either due to aselective defect in secretion, or impaired matrix assembly. Our

observation that laminin-1 assembly around Sec13-suppressedcysts is impaired points to an additional defect in matrixremodelling. Rac1-dependent remodelling of the lamininnetwork has been shown to be essential for cyst morphogenesis

in other systems (O’Brien et al., 2001). It is also possible that thedefect in Sec13–Sec31 coupling reduces secretion of other keymacromolecules (or molecules) that are required but not involved

in ECM assembly.

We also found perlecan levels to be reduced and this couldcontribute to the phenotype because perlecan crosslinks several

matrix components and, hence, is likely to be involved incollagen and/or laminin assembly in Sec13-depleted cysts.However, if knockdown of Sec13 in zebrafish results in a

general defect of perlecan secretion one would expect to largelyreproduce the phenotype of a knockout of perlecan in mice (Rossiet al., 2003). For example, knockout of perlecan was reported tohave a major affect on the lens capsule and we never observed

any effects on lens integrity in Sec13 morphants (Townley et al.,2008) (K.S. and D.J.S., unpublished observations). We thereforeassume that a defect of perlecan secretion is unlikely to play a

major causative part in the failure of tube formation in Sec13-depleted Caco-2 cells.

It has been well documented that the ECM dictates the

orientation of the spindle axis during mitosis in a variety ofsystems (Bornens, 2008). Caco-2 cyst morphogenesis requiresCdc42-dependent signalling to specify the alignment of the mitotic

Fig. 6. Cyst morphogenesis defects in 3D matrix following Sec13

suppression can be substantially rescued by co-culture with human

fibroblasts. (A) Lumen formation is partially restored in cysts formed from

Sec13-suppressed cells in the presence of fibroblasts. Cysts are more

spherical, showing well-defined apical actin organization. Cells are still seen

within the cyst in addition to the limiting monolayer, suggesting a partial but

incomplete recovery of morphogenesis. Scale bar: 20 mm. (B) Quantification

of cyst lumen area from three independent experiments shows that growth in

the presence of fibroblasts restored lumen expansion to Sec13-1 depleted

cysts. Similar results were obtained for Sec13-2 cells. Boxes show the median

with 25th and 75th percentiles; whiskers show the spread of the data. n.20

(typically 20–30) cysts in each of three independent experiments performed

for each experimental condition. Results of statistical testing (Student’s t-test)

compared with controls in 3D matrix alone are indicated.

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spindle and this results in subsequent positioning of the apical

surface during cyst expansion (Jaffe et al., 2008). We noticed a

significant misalignment of mitotic spindles in cysts grown from

Sec13-suppressed cells. Our interpretation of the data is that COPII-

dependent secretion (including secretion of ECM components) and

subsequent remodelling of laminin-rich ECM is secondarily

required for Cdc42 activation, consistent with work showing that

outside-in signalling by laminin-1 is required for Cdc42 activation

in neurite outgrowth (Weston et al., 2000).

Caco-2 cells (and other epithelial lines) in both 2D and 3D

culture are widely used in basic biomedical science but also

in the pharmaceutical industry. An important outcome of these

experiments is the demonstration that epithelial cells, when

embedded in a 3D matrix, retain an essential requirement for

Fig. 7. Rescue of Sec13 lumen defect by addition of

fibroblast-conditioned medium. Caco-2 cells expressing

scrambled shRNA or Sec13 shRNA were embedded in

Geltrex and grown for 7 days. Cells were grown in the

presence of either control (Caco-2) medium or conditioned

medium taken from a confluent dish of NDF. (A) Scramble

shRNA-transfected Caco-2 cells plus Caco-2 medium.

(B) Scramble shRNA-transfected Caco-2 cells plus NDF

medium. (C) Sec13 shRNA-transfected Caco-2 cells plus

Caco-2 medium. (D) Sec13 shRNA-transfected Caco-2

cells plus NDF medium. (E–H) Representation of mean

lumen area for cysts formed in A–D; green represents

basolateral surface with the apical surface in red.

(I) Quantification of mean lumen size for cysts in A–D;

median values are shown by the horizontal bar within each

box; boxes show 25th and 75th percentiles; whiskers show

the spread of the data. n.20 cysts in each of three

independent experiments performed for each experimental

condition. Scale bars: 10 mm.

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COPII-dependent secretion in order to fully differentiate into anepithelial cyst. Given the conservation of membrane trafficking

between species, this is likely to be true for other widely used modelsystems, including Madin-Darby canine kidney cells and mouse

mammary gland epithelia. The necessity for ongoing secretionby Caco-2 cells in 3D culture has key implications for theinterpretation of any work using 3D cell culture models. Our data

show that exogenously supplied EHS-derived matrix alone is notsufficient to facilitate proper cyst morphogenesis. Experimental

outcomes that show clear defects in epithelial organization andmorphogenesis could result from secondarily impaired ECMsecretion rather than only from primary effects on intracellular

signalling pathways. Taken together, the data we present here showthat efficient COPII assembly and function is indispensable for

completion of epithelial and cyst morphogenesis. The exact natureof late COPII function and potentially affected cargo molecules willrequire further research. Our results have significant implications

for COPII function, for epithelial polarity and organization in vivo,and for the interpretation of experiments using Caco-2 cells

embedded in EHS-derived tumour matrix.

Materials and MethodsAll reagents were purchased from Sigma-Aldrich (Poole, UK) unless otherwiseindicated.

Growth of cultured cells.

Caco-2 cells were maintained in DMEM supplemented with 10% FCS (Invitrogen,Paisley, UK), 1% glutamine and 1% non-essential amino acids. Primary humandermal fibroblasts were purchased from Cascade Biologics (Invitrogen) and werecultured in Medium 106 supplemented with low-serum growth supplement. At24 hours prior to the start of the experiments, cells were seeded onto either 22-mmcoverslips, glass-bottom dishes (MatTek, Ashland, MA) or polyester transwellpermeable supports (Corning).

Antibodies and other reagents

Monoclonal mouse anti-human collagen IV (MAB1910, anti-collagen type IV a2chain, clone 23IIC3) was from Millipore (Watford, UK); polyclonal anti-laminin(ab11575) was from Abcam (Cambridge, UK); and rat monoclonal anti-heparansulphate proteoglycan (Perlecan, MAB1948, clone number A7L6) was fromMillipore. The following antibodies were used to detect laminin: laminin c2 (SC-7652; Santa Cruz Biotechnology, Santa Cruz, CA), laminin b3 (SC-20775; SantaCruz Biotechnology) and rabbit polyclonal to laminin (which recognises A chain, B1chain and B2 chain) (ab11575; Abcam, Cambridge, UK). Anti-b-catenin (C19220-050, clone number 14) was from BD Transduction Laboratories (Franklin Lakes,NJ). Anti-tubulin (MS-581-P0, clone DM1A) was from Neomarkers (Fremont, CA).Sec13 antibodies were a generous gift from Wanjin Hong (IMCB, Singapore) andBeatriz Fontoura (Southwestern Medical Center, Dallas, TX). Anti-gp135/podocalyxin was kindly provided by Paul Verkade (University of Bristol, Bristol,UK). Anti-Sec31A antibodies were raised against synthetic peptides synthesized byGraham Bloomberg, University of Bristol and coupled to KLH before immunizationinto rabbits. Antibodies were affinity purified using peptide coupled to sulfolinkresin (Pierce, Cramlington, UK) using the manufacturer’s protocols. The peptidesequence was MKLKEVRTAMQAWS(C), C-terminal cysteines were added forcoupling. Mouse monoclonal ESA was a kind gift from Andre Le Bivic (IBDM,Marseilles, France). Secondary antibodies were from Jackson ImmunoResearchLaboratories (West Grove, PA). Alexa-Fluor-568-labelled phalloidin was fromMolecular Probes.

Lentiviral constructs and generation of stably suppressed cell lines

Short hairpin RNA (shRNA) lentiviral particles were from Dharmacon (ThermoScientific). The sequences targeted were 59-GCCTTAACGTGATCGGAGA-39 forSec13-1 and 59-ATGAGGACATGATTCACGA-39 for Sec13-2. Caco-2 cells wereplated at 1.56105 cells per 3-cm tissue culture dish and infected with lentivirusparticles according to the manufacturers’ instructions. Following puromycinselection, stable cell lines were verified by western blot and immunofluorescence.Controls were infected with a scrambled shRNA lentivirus.

Growth of Caco-2 cells in 2D and 3D

Caco-2 stable cell lines were grown on permeable supports to allow polarization.Polyester transwell permeable supports, 0.4 mm thick and 12 mm diameter werefrom Corning. Cells were seeded at a density of 26105 cells per insert and

cultured for 2 weeks with media changes every other day. The cells were then

either processed for immunofluorescence or electron microscopy. For growth in

3D, stable cell lines were seeded into Geltrex (Invitrogen) to allow polarizationinto 3D cysts. All experiments shown were performed using a single lot number.

Cells were trypsinized and counted, and then 66104 cells per well in an eight-well

chamber slide were mixed with Geltrex to give a final concentration of 40%. A

sample of 100 ml was plated into the chamber slide well and incubated at 37 C for

30 minutes to allow the Geltrex to solidify. Fresh medium (400 ml) was then added

on top. Cells were cultured for 3 days or 7 days with media changes every otherday.

Cell-derived matrices

Culture dishes were prepared before seeding with live cells by first washing withphosphate-buffered saline (PBS) and incubating with 0.2% sterile gelatin for

60 minutes at 37 C (2% gelatin, Sigma G1393, diluted in PBS). They were then

washed three times with PBS and crosslinked with 1% sterile glutaraldehyde in PBS

for 30 minutes at room temperature. The dishes were washed three times with PBS

before quenching the crosslinker with 1 M sterile glycine in PBS for 20 minutes atroom temperature. The dishes were then washed three times with PBS, followed by

incubation with DMEM growth medium for 30 minutes at 37 C. Caco-2 cells were

trypsinized and counted using a hemocytometer; 16105 cells/ml were plated and

2 ml was used per live cell dish. Cells were cultured at 37 C, 5% CO2 overnight on

the gelatin-coated dishes. The cells must be confluent throughout the generation

of the matrix so, once confluent, the cells were fed with complete mediumsupplemented with 50 mg/ml ascorbic acid. The cells were cultured for 8 days with

media changes every day using medium supplemented with 50 mg/ml ascorbic acid.

To denude the cells, the medium was first removed and cells washed with PBS.

Then, 1.5 ml of pre-warmed extraction buffer (20 mM NH4OH and 0.5% Triton X-

100 in PBS) was gently added. Cells were lysed for 2 minutes until no intact cells

were visible. The extraction buffer was aspirated and washed twice with PBScontaining calcium and magnesium. DNA residue was digested by incubating with

10 mg/ml DNase I in PBS containing calcium and magnesium for 30 minutes at

37 C, 5% CO2. This was removed and the dishes were washed with PBS containing

calcium and magnesium. The matrices were then processed for immunofluorescence

or immunoblotting. For immunofluorescence, labelling with collagen IV or laminin

antibodies was carried out for 2 hours at 37 C, 5% CO2, before fixation. Otherprimary antibodies were incubated for 2 hours at room temperature after fixation

with paraformaldehyde. For immunoblotting, matrix was solubilized in SDS-PAGE

sample buffer prior to electrophoresis and immunoblotting with antibodies.

Co-culturing Caco-2 cells with normal dermal fibroblasts

Stable Caco-2 cell lines were seeded with normal human fibroblasts into Geltrex to try

to rescue the lumen expansion defect. Caco-2 cells and fibroblasts were trypsinized

and counted, and then 66104 Caco-2 cells and 36104 fibroblasts per well of an eight-

well chamber slide were mixed with Geltrex to give a final concentration of 40%.Then, 100 ml was plated into each well and put at 37 C for the Geltrex to solidify;

400 ml of Caco-2 medium was then put on top. For the conditioned medium

experiment, 200 ml of Caco-2 medium and 200 ml of medium taken from a confluent

dish of normal dermal fibroblasts was added. Medium was changed every 2 days and

the cells were grown for 7 days to allow the formation of cysts.

Danio rerio morpholino oligonucleotide microinjection

Husbandry of zebrafish (D. rerio) AB strain and morpholino injection of was

performed as described previously (Townley et al., 2008). Two translation-blocking morpholino oligonucleotides (Sec13-1 and Sec13-2) were validated

previously (Townley et al., 2008) but for the experiments presented in Fig. 1 only

that targeting Sec13-2 was used.

Electron microscopy

Sec13- or lamin-A/C (control)-suppressed Caco-2 cells were fixed in 2.5%

glutaraldehyde containing 0.1 M cacodylate for 2 hours before transwell filters

were cut. Morpholino-injected fish (5 dpf, days post fertilization) were fixed in 5%

glutaraldehyde, 0.05 M cacodylate, 1% paraformaldehyde, 1% sucrose and 1 mMMgCl2 for 2 hours. Samples were post-fixed in 1% (for cells) or 2% (for zebrafish

embryos) OsO4, respectively, and dehydrated through a graded series of ethanol.

Zebrafish and cells were infiltrated with Epon and eventually embedded in moulds.

The Epon was hardened for 48 hours and 50 nm sections were counterstained and

analyzed with a FEI Tecnai12 Biotwin equipped with a bottom-mount 4K EAGLE

CCD camera. For histology, 1 mm Epon sections of fish from the same anterior–posterior region were counterstained with Methylene Blue, mounted and imaged

on a Zeiss Axioplan 2 microscope with a Qimaging digital camera.

For SEM, shRNA-expressing Caco-2 cells were fixed in 2.5% glutaraldehydecontaining 0.1 M cacodylate, then post-fixed in 1% OsO4 in cacodylate for 1 hour,

dehydrated in graded ethanol, and critical-point dried (100% ethanol, carbon

dioxide). The specimens were sputter-coated with gold (Edwards Sputter Coater)

and viewed on a FEI Quanta4000 scanning electron microscope.

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Immunolabelling

Medium was removed from the cysts and they were subsequently washed withPBS. Cysts were then fixed using 4% paraformaldehyde, for 30 minutes at roomtemperature. This was removed and the cysts incubated with 0.5% Triton X-100 inPBS for 30 minutes at room temperature. This was followed by incubation at roomtemperature with 100 mM glycine in PBS for 10 minutes. Cysts were then blockedusing 3% bovine serum albumin (BSA) in PBS for 1 hour at room temperature.Primary antibodies were then incubated with the cysts overnight at 4 C. Primaryantibodies were diluted to between 1:100 and 1:200 in 3% BSA in PBS. Followingincubation with primary antibodies, cysts were washed with PBS for 5 minutes andthis was repeated three times. Secondary antibodies, diluted in 3% BSA in PBS,were then added and incubated for 2 hours at room temperature. Cysts werewashed in PBS for 10 minutes and this was repeated three times. Nuclear stainingwas achieved by counterstaining cysts with DAPI for 15 minutes at roomtemperature. Cysts were then washed in PBS and then stored in PBS at 4 C untilthey were imaged.

Confocal imaging

Cysts cultured in chamber slides and processed for immunofluorescence wereimaged using a Leica AOBS SP2 confocal imaging system attached to a LeicaDMIRE2 inverted microscope (Leica Microsystems, Milton Keynes, UK).Confocal Z slices of 0.8 mm were taken through the cyst using a 405 nm diodelaser, a 488 nm argon laser, and 543 nm and 633 nm red HeNe lasers. Data wasprocessed using Volocity software (Perkin Elmer) and Adobe Illustrator CS(Adobe).

Quantification of image data and statistical analysis

Analysis of spindle angle was performed as described (Jaffe et al., 2008). Analysisof lumen size and number of nuclei was performed using the measurement tools inVolocity 5.1 (Perkin Elmer). Intensity and area measurements for ECM werecompared using automated object identification and counting using Volocity 5.1.Samples were compared using a Student’s 1-tailed unpaired t-test using GraphPadPrism. Note that graphs show box and whisker plots from GraphPad, where boxesindicate the 25th and 75th percentiles and the whiskers display the spread of thedata.

AcknowledgementsThe authors wish to thank Harry Mellor, Mark Bass, Paul Martin,Paul Verkade and Jo Adams for very helpful discussions and criticalreading of the manuscript. We acknowledge the kind gifts ofantibodies from Andre le Bivic and Paul Verkade, and thank Yi Fengand Paul Martin for help with the zebrafish work, and Ginny Tilly forassistance with electron microscopy. We are also indebted to KateHeesom for performing 2D gel electrophoresis.

FundingThis work was funded through an MRC Non-Clinical SeniorFellowship to D.J.S. [grant number G117/553] and a BBSRC grant[grant number E019633]. Deposited in PMC for release after 6months.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.091355/-/DC1

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